Technical Field
The present disclosure relates to novel, small molecule inhibitors of gene transcription mediated by myocardin-related transcription factor and/or serum response factor (“MRTF/SRF”), and methods of using the small molecules to inhibit MRTF/SRF-mediated gene transcription and to treat diseases.
Description of Related Technology
Cell growth, proliferation, migration, and invasion are dependent on many growth factors, mitogens, and chemotactic agents. The medium for growing cells in tissue culture generally contains serum (e.g., fetal bovine serum) and serum also stimulates migration and invasion of cancer cells and fibroblasts. Treatment of cells with serum results in robust activation of gene transcription via the serum response factor (“SRF”) (see Norman et al., Cell 55:989-1003 (1988)). SRF is associated with cellular transformation and epithelial-mesenchymal transformation (see Iwahara et al., Oncogene 22:5946-5957 (2003); Psichari et al., J Biol Chem 277:29490-29495 (2002)).
One key mechanism of activation of SRF by serum involves activation of the Rho GTPases (especially rhoA and rhoC) through G protein-coupled receptors and possibly other mechanisms. Activation of rhoA and rhoC induces actin polymerization and release of the transcriptional coactivator, myocardin-related transcription factor (“MRTF”) (see Cen et al., Mol Cell Biol 23:6597-6608 (2003); Miralles et al., Cell 113:329-342 (2003); Selvaraj and Prywes, J Biol Chem 278:41977-41987 (2003)). MRTF, which is also known as MKL, was first identified as a site of gene translocation in leukemia (megakaryoblastic leukemia), like the leukemia-associated rhoGEF (“LARG”, see Mercher et al., Genes Chromosomes Cancer 33:22-28 (2002)). The protein product of the translocated gene is hyperactive compared to the wild-type protein. MRTF and MKL have also been called modified in acute leukemia (“MAL”) and BSAC (see Miralles et al., Cell 113:329-342 (2003); Sasazuki et al., J Biol Chem 277:28853-28860 (2002)). There are two MRTF genes (MRTF-A and MRTF-B or MKL1 and MKL2, respectively). Their actions are largely redundant where both proteins are expressed (see Shaposhnikov, Cell Cycle 12:1762-72 (2013)). MRTF was identified in an antiapoptosis screen for genes that abrogate tumor necrosis factor-induced cell death (see Sasazuki et al., J Biol Chem 277:28853-28860 (2002)). As a consequence of rho signaling, MRTF translocates to the nucleus and binds SRF leading to the expression of c-fos which, along with c-jun, forms the transcription factor AP-1. The AP-1 transcription factor promotes the activity of various MMPs and other cell motility genes (see Benbow and Brinckerhoff, Matrix Biol 15:519-526 (1997)). Expression of these genes leads to cancer cell invasion and metastasis. Thus, there is a link between Rho/MRTF-controlled biological processes and cancer metastasis. Similarly, both LARG and MKL are important players in these processes.
Rho GTPase signaling and MRTF-regulated gene transcription have also been implicated in tissue fibrosis in lung (see Sandbo et al, Am J Respir Cell Mol Biol. 41:332-8 (2009); Luchsinger J Biol Chem. 286:44116-25 (2011)), skin (see Haak et al, J Pharmacol Exp Ther. 349:480-6 (2014)), and intestine (see Johnson et al, Inflamm Bowel Dis. 20:154-65 (2014)). Many genes involved in fibrosis (alpha-smooth muscle actin, CTGF, and collagen itself) are activated by Rho-regulated MRTF/SRF mechanisms (see Haak et al, J Pharmacol Exp Ther. 349:480-6, (2014)).
Cancer metastasis is a significant medical problem in the United States, where it is estimated that over 500,000 cancer-related deaths in 2003 resulted from metastatic tumors rather than primary tumors (approximately 90% of cancer deaths). Cancer metastasis requires malfunction in several tightly regulated cellular processes controlling cell movement from a primary site to a secondary site. These cellular processes include cell survival, adhesion, migration, and proteolysis resulting in extracellular matrix remodeling, immune escape, angiogenesis and lymphangiogenesis, and target ‘homing’ Most existing cancer treatments focus on killing tumor cells; however, such chemotherapeutic intervention leads to substantial toxicity to healthy cells and tissue. Since spread, or metastasis, of cancers is the primary cause of cancer-related mortalities, there is a need for agents that can specifically inhibit or prevent signals that trigger metastasis.
Rho proteins are overexpressed in various tumors, including colon, breast, lung, testicular germ cell, and head and neck squamous-cell carcinoma (see Sawyer, Expert Opin. Investig. Drugs., 13: 1-9 (2004)). The rho family of small GTP binding proteins plays important roles in many normal biological processes and in cancer (see Schmidt and Hall, Genes Dev., 16:1587-1609 (2002); Burridge and Wennerberg, Cell, 116:167-179 (2004)). This family includes three main groups: rho, rac, and cdc42. Rho is activated by numerous external stimuli including growth factor receptors, immune receptors, cell adhesion, and G protein coupled receptors (GPCRs) (see Schmidt and Hall, Genes Dev., 16:1587-1609 (2002), Sah et al., Annu. Rev. Pharmacol. Toxicol., 40:459-489 (2000)).
RhoA and rhoC play roles in metastasis (see Clark et al., Nature 406:532-535 (2000); Ikoma et al., Clin Cancer Res 10:1192-1200 (2004); Shikada et al., Clin Cancer Res 9:5282-5286 (2003); Wu et al., Breast Cancer Res Treat 84:3-12 (2004); Hakem et al, Genes Dev 19:1974-9 (2005). Both rhoA and racl can regulate the function of the extracellular matrix (ECM) proteins, ezrin, moesin, and radixin, by the phosphorylation of ezrin via the rhoA pathway and the phosphorylation of the ezrin antagonist, neurofibromatosis 2, by the racl pathway (see Shaw et al., Dev Cell 1:63-72 (2001); Matsui et al., J Cell Biol 140:647-657 (1998)). These ECM proteins promote cell movement by utilizing the ECM receptor, CD44, to link the actin cytoskeleton with the plasma membrane. In addition, rhoA and racl regulate ECM remodeling by controlling the levels of matrix metalloproteinases (MMPs) or their antagonists, tissue inhibitors of metalloproteinases (TIMPs) (see Bartolome et al., Cancer Res 64:2534-2543 (2004)). RhoA is also required for monocyte tail retraction during transendothelial migration, indicating a role in extravasation, which is a key process in metastasis (see Worthylake et al., J Cell Biol 154:147-160 (2001).
The relative contributions of rho and rac proteins in the metastatic phenotype has been studied (see Sahai and Marshall, Nat Rev Cancer 2:133-142 (2002); Whitehead et al., Oncogene 20:1547-1555 (2001)). Sahai and Marshall (see Nat Cell Biol 5:711-719 (2003)) showed that different tumor cell lines exhibit different mechanisms of motility and invasion. In particular, 375 m2 melanoma and LS174T colon carcinoma cell lines showed striking “rounded” and “blebbed” morphology during invasion into Matrigel matrices. This invasion was entirely rho-dependent and was blocked by C3 exotoxin, the N17rho dominant negative protein, and a ROCK kinase inhibitor. In contrast, two other cell lines were blocked instead by a rac dominant negative mutation, but not rho or ROCK inhibitors. These latter two cell lines (BE colon carcinoma and SW962 squamous cell carcinoma) had elongated morphologies. A third line showed a mixed morphology and was blocked partially by both rho and rac inhibitors. Additionally, mice lacking rhoC have greatly reduced metastasis of virally-induced breast tumors to lung (see Hakem et al, Genes Dev 19:1974-9 (2005)). Also, knock-down of SRF or its transcriptional co-activator MKL reduced lung metastases from breast or melanoma xenografts (see Medjkane et al, Nat Cell Biol. 11:257-68 (2009)). Thus, there is important heterogeneity in mechanisms of tumor cell behavior that contributes to metastasis. It is widely recognized that cell growth and apoptosis mechanisms vary greatly among tumors, necessitating customized therapeutic approaches.
Nearly 40% of chronic diseases such as cirrhosis, heart failure, and diabetic nephropathy are characterized by fibrosis or excess deposition of extracellular matrix, including collagen. The poor clinical outcome of several orphan diseases (scleroderma or systemic sclerosis (“SSc”), idiopathic pulmonary fibrosis (“IPF”) etc.) is primarily determined by tissue fibrosis; there are absolutely no effective treatments despite their rapid and lethal clinical course.
Systemic sclerosis is an orphan, multisystem autoimmune disorder that can cause fibrosis of the skin and internal organ systems (lungs, heart, kidneys, and gastrointestinal system). It has the highest case fatality rate of any rheumatic disease. SSc predominately affects women (see Beyer et al., Arthritis Rheum 62: 2831-2844 (2010); Boukhalfa G, et al., Exp Nephrol 4: 241-247 (1996); Buhl A M, et al., J Biol Chem 270: 24631-24634 (1995); Chaqour et al., FEBS J 273: 3639-3649 (2006); Charles et al., Lancet 367: 1683-1691 (2006) and increases with age. The precise pathogenesis of SSc is yet to be defined but the major clinical features of SSc—collagen production, vascular damage and inflammation/autoimmunity—require environmental triggers and genetic effects which interact with the three cardinal features of the disease at several points (see Charles et al., Lancet 367: 1683-1691 (2006)). Generally, there is initial inflammation but fibrosis persists even after the inflammation has resolved or has been suppressed by medications (see Beyer et al., Curr Opin Rheumatol 24: 274-280 (2012); Wynn T A, and Ramalingam T R. Nat Med 18: 1028-1040 (2012)).
Therefore, there is a need for new compounds and methods for targeted therapy that can treat and manage cancer and fibrosis.